Polyphosphate (polyP) is a linear polymer of inorganic phosphates that is secreted by activated platelets and is abundant in many pathogenic microorganisms. Our lab has recently shown that polyP from bacteria and human platelets play important roles in inflammation and coagulation in vitro and in vivo, supporting the paradigm of polyP as the long-sought (patho)physiologic activator of the contact pathway of blood clotting. These studies also implicate polyP in contributing to thrombosis and consumptive coagulopathies accompanying bacterial sepsis. PolyP polymer lengths are known to vary substantially among different organisms and cell types, with shorter polymers secreted by human platelets and much longer polymers accumulating in microorganisms. Our preliminary data demonstrate that long polyP polymers (synthetic and bacterial) exert differential effects on blood clotting compared to the shorter polyP polymers secreted by platelets, raising intriguing questions about the mechanisms by which various polyP sizes differentially modulate the blood clotting system. The long-term goal of this proposal is to advance our understanding of the mechanisms by which polyP modulates blood clotting and inflammation, with a particular emphasis on the contact pathway. To accomplish this goal, the first specific aim is to use bacterial and platelet-derived polyP, in addition to carefully size-fractionated polyP preparations, to investigate how polyP promotes each of the individual enzyme reactions that result in triggering the contact pathway. Understanding how polyP of varying polymer lengths modulate the contact phase of blood clotting will shed new light on the (patho)physiologic roles of this pathway. We have recently developed the method of covalently immobilizing polyP onto a variety of solid supports. This technological advancement and the potent procoagulant activities of polyP support the second aim to develop novel, local-acting hemostatic agents to control hemorrhage. We will employ the newly developed method for manipulating polyP to covalently attach polyP onto biomaterial surfaces such as collagen-based wound dressings. The third aim will identify proteins and small compounds that can inhibit the procoagulant and proinflammatory activities of polyP. We will investigate the abilities of isolated polyP-binding domains from E. coli Lon protease and exopolyphosphatase, as well as small cationic compounds, to inhibit polyP procoagulant activity in vitro. We will also engineer polyP-binding proteins with enhanced affinity for polyP for the eventual use of polyP-binding molecules to abrogate the prothrombotic and proinflammatory activities of polyP in vivo. Results from the studies in these three aims will provide new insights into how polyP modulates the blood clotting system to drive thrombosis and inflammation.